Quantitative intracellular calcium imaging with laser-scanning confocal microscopy.
Laser-scanning confocal microscopy has been used to visualise the fluorescence of a visible wavelength Ca2(+)-sensitive fluorophore, Fluo-3 in isolated cardiac myocytes. A protocol for the derivation of quantitative information from this single wavelength indicator is presented. This paradigm involves co-loading cells with two Ca2(+)-sensitive fluorescent indicators, Fluo-3 and Fura-2. Wide-field ratiometric measurements of Fura-2 fluorescence provided a baseline [Ca2+] upon which changes in Fluo-3 fluorescence could be directly expressed as [Ca2+] changes. The Ca2+ changes occurring in spontaneously active cardiac cells are presented as an example of the method. Although fluorescence energy transfer between Fura-2 and Fluo-3 was detectable in some in vitro mixtures of the two fluorophores, this process was not evident in co-loaded cardiac cells under the loading conditions employed.
Williams
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DA
Department of Physiology, University of Melbourne, Parkville, Victoria, Australia.
D000814:Aniline Compounds; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2; D007834:Lasers; D008856:Microscopy, Fluorescence; D009200:Myocardial Contraction; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D014966:Xanthenes
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D014966:Xanthenes; C059715:Fluo-3; D002118:Calcium; D016257:Fura-2